Bisulfite-Based Methylation Analysis at the Maize Flowering Time Locus Vgt1

One of the major quantitative trait locus (QTL) for flowering time in maize, the Vegetative to generative transition 1 (Vgt1) locus, corresponds to an upstream (70 kb) non-coding regulatory element of ZmRap2.7, a repressor of flowering. Among the polymorphisms that distinguish late and early Vgt1 alleles, the insertion of a MITE transposon was found to be highly associated with flowering time in independent studies. As cytosine methylation is known to influence gene expression, we aimed to determine if methylation might be involved in the relationship existing between Vgt1 and Rap2.7. The methylation state at Vgt1 was assayed using an approach that combines digestion with McrBc (an endonuclease that acts upon methylated DNA), and quantitative PCR. The analyses were performed on genomic DNA from leaves of six different maize lines at four stages of development. The results showed a trend of reduction of methylation from the first to the last stage with the exception of a short genomic region flanking the MITE insertion, which showed a constant and very dense methylation throughout leaf development and for both alleles. Bisulfite sequencing of a small portion of Vgt1 revealed differential methylation for a single cytosine between the two alleles. Finally, ZmRap2.7 expression was determined at four developmental stages for the six genotypes and found to correlate with ZmRap2.7 transcription.